Optimal soaking conditions and addition of exogenous substances improve accumulation of γ-aminobutyric acid (GABA) in germinated mung bean (Vigna radiata)

Germinated mung beans have been widely used as fresh vegetable or processed healthy food due to their high amounts of bioactive and nutritional compounds, including γ-aminobutyric acid (GABA). The objectives of this study were to investigate the effects of different soaking conditions (temperature, ratios of seeds to water, soaking time, pH, addition of L-glutamic acid (L-Glu) or gibberellic acid (GA3)) and germination time on accumulation of GABA in the germinated mung bean seeds. The mung bean seeds, soaked at 40°C for 4 h with ratio of seeds to water of 1:4 (g mL⁻¹) and then germinated for 7 h, accumulated higher amount of GABA than other soaking conditions and germination time. The addition of GA3 (0.30 mg L⁻¹) or L-glu (1,000 mg L⁻¹) or acidifying to pH 5.5 of the soaking water had high impact on the GABA accumulation. Among them, the soaking water with pH 5.5 was more effective than adding with the L-Glu or GA3 in the production of GABA (1677 mg/kg powder) and essential amino acids (16.56 g/100g powder) in the germinated mung bean seeds. The findings of this study provide useful information to produce GABA-enriched and healthy foods from mung beans.     

Effects of soaking and aeration treatment on γ-aminobutyric acid accumulation in germinated soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.)

Effects of soaking temperature, soaking time, opportunity of aeration treatment, culture temperature, pH value, and air flow rate on γ-aminobutyric acid (GABA) accumulation during germination of soybean (Glycine max (L.) Merr.) were investigated in this study. The objective of this study was to optimize the culture conditions of GABA production in germinated soybeans. Results showed that soaking at 30 °C for 4 h was found to be the most effective soaking ways that made soybean seeds have sufficient moisture, satisfactory germination, and GABA accumulation. The suitable stress opportunity was dark culture for 2 days with distilled water and then hypoxia stress in aerated culture medium for 2 days in a dark incubator at 30 °C. Under these conditions, the maximum GABA content (2.24 mg/g DW) was 3.5 times higher than the initial sample of aeration treatment (0 h) and 12.5 times higher than the raw material. Correlation analysis also revealed that GABA accumulation was significantly correlated to the corresponding physiochemical indexes (p < 0.01). Box-Behnken experimental analysis showed that the optimal condition with aeration treatment for GABA accumulation in germinated soybean was at a temperature of 30.5 °C, a pH value of 4.1, and an air flow rate of 0.9 L/min; the predicted highest GABA yield was 2.60 mg/g DW, which was 15.2 times higher than raw seeds. Analysis of variance and confirmatory trials for the regression model suggested that the model can quite exactly predict GABA accumulation in soybean during germination.     

Effects of aminoguanidine on γ-aminobutyric acid accumulation pathways in fresh tea leaves under hypoxic conditions

Aminoguanidine (AG), a specific inhibitor of diamine oxidase (DAO), can be used to investigate the γ-aminobutyric acid (GABA) accumulation pathways. The effect of AG on GABA accumulation pathways of tea fresh leaves remains to be explored. Our results indicated AG concentration did not significantly affect GAD activity, but high concentration solution made GABA-T activity increase. AG (1 mm ) maximally, but not entirely, blocked the polyamine degradation pathway, but had no effect on GAD and GABA-T. The polyamine degradation pathway was responsible for 26.8% and 17.0% of GABA formation under hypoxic conditions involving immersion and immersion–vacuum, respectively, because DAO and PAO activity was not completely inhibited.     

Dynamics of γ-aminobutyric acid in wheat flour bread making

The dynamics of the health-improving non-protein amino acid γ-aminobutyric acid (GABA) during bread making were studied. Wheat flour contains trace levels of GABA (<15 ppm) and ca. 160–175 ppm of its precursor, glutamic acid (GA). During dough mixing, the levels of both GA and GABA largely increased. While wheat flour endogenous glutamic acid decarboxylase (GAD) performs some minor conversion of GA into GABA, yeast is the main contributor to GABA formation. Comparison of amino acid levels of dough samples, without or with yeast, indicated that yeast favours both GA and GABA formation already during mixing. Fermentation decreased both GA and GABA contents, due to amino acid consumption by the yeast, which used more GA than GABA. Proofing and baking resulted in large GABA losses, the latter probably in Maillard browning reactions during baking. Thermal loss of GA was less pronounced than that of GABA. Breads contained only trace levels of GABA and ca. 90–130 ppm of its precursor. Exogenous supplementation of recombinantly produced GAD of Yersinia intermedia decreased GA levels in mixed and fermented dough and increased GABA levels. The highest GAD dosage used resulted in fermented doughs with ca. 300 ppm of GABA, i.e. three times higher than the level present in the reference sample (no GAD added). After baking, a significant GABA level was left in the bread samples (ca. 115 ppm) and GABA-enriched breads were obtained. Addition of sodium glutamate (100–380 ppm) to a bread recipe containing no added GAD clearly indicated that its precursor was not the limiting factor for GABA conversion during bread making since the resulting breads contained no GABA, or only trace levels (ca. 20 ppm).     

In situ production of γ-aminobutyric acid in breakfast cereals

Breakfast cereals are an important part of an equilibrated diet in the Western world, making them extremely suited for carrying health benefits. Intake of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter of the nervous system, has been related to blood pressure lowering in hypertensive individuals. In vivo, GABA is formed from glutamic acid (GA) by glutamic acid decarboxylase (GAD), a widely distributed enzyme in prokaryotic and eukaryotic species. We here enriched breakfast cereals with GABA by recipe and process optimisation. The dynamics of GA and GABA were monitored throughout the production process. Addition of exogenous recombinantly produced GAD of Yersinia intermedia increased GABA levels by 2- to 5-fold. As only trace levels of GABA (<15 ppm) and relatively low levels of its precursor (GA, <100 ppm) are present in the wheat and rice flour used, a well-thought ingredient choice (inclusion of quinoa flour (ca. 90 ppm GABA and 700 ppm GA) or bran enrichment (ca. 66 ppm GABA and 500 ppm GA)) also significantly increases the GABA content in the final flakes. Finally, a strict control of the heating steps during the production process reduces GA and GABA losses. Consumption of one portion (30 g) of the here produced enriched breakfast cereals can even meet up to 55% of the daily intake earlier reported to lower blood pressure (ca. 10 mg).     

Influence of preprocessing methods and fermentation of adzuki beans on γ-aminobutyric acid (GABA) accumulation by lactic acid bacteria

The effects of pre-processes (immersing, germinating, and cold shock) and fermentation conditions of adzuki beans on γ-aminobutyric acid (GABA) accumulation using mixed cultures of Lactococcus lactis and Lactobacillus rhamnosus were investigated in this study. Among the preprocessing methods, cold shock treatment resulted in the highest observed GABA content (201.2 mg/100 g); a 150-fold increase compared to the non-treated adzuki beans. The LAB strains grew rapidly in cold-treated adzuki bean substrates and reached 10⁸ cfu/ml after 24 h of fermentation at 30 °C. After optimization, the GABA yield reached 68.2 mg/100 ml; a 20-fold increase compared to the non-fermentation yield. The viable cell counts of LAB remained above 10⁸ cfu/ml after 28 days of storage at 4 °C. Our results suggest that the combination of cold shock pretreatment and fermentation by LAB may be used for the preparation of adzuki beans with high GABA content, which can then be used as a natural resource of functional foods.     

Effects of heating method and temperature in combination with hypoxic treatment on γ-aminobutyric acid,phenolics content and antioxidant activity of germinated rice

Heat and hypoxic treatments were combined with soaking to enhance γ-aminobutyric acid (GABA) production in germinated paddy (GP). Effects of heating temperature, heating techniques, that is, impinging stream drying (ISD) and tray drying (TD), and germination time on the GABA content, phenolics content and antioxidant activity were also investigated. Heating temperature had a significant effect on the GABA content; both ISD and TD helped increase the GABA content when the post-treatment grain temperature was 38–40.5 °C. Within this grain temperature range, soaking in combination with heat and hypoxic treatments (S-ISD-H and S-TD-H) yielded GP with higher GABA content than soaking (S) and soaking in combination with hypoxic treatment (S-H). However, ISD was noted to be more suitable than TD. Phenolics content and antioxidant activity of GP prepared by S-ISD-H at different heating temperatures and S-H were not significantly different but were higher than those of the GP prepared by S.     

Influence of processing on the generation of γ-aminobutyric acid in green coffee beans

A determination of the concentrations of free amino acids in differently processed green coffees indicated the nonprotein amino acid -aminobutyric acid (GABA), a well-known plant stress metabolite, to be present in raw coffee beans (Coffea arabica L.) in significantly varying amounts. The GABA content of unwashed Arabica beans (green coffee produced by the dry processing method) was always markedly higher than that of washed Arabicas (wet processing method) as well as that of untreated seeds. This result underlined the assumption that during postharvest treatment a significant metabolism occurs within coffee seeds. A putative relation between drought stress of the coffee seeds and postharvest treatment methods is discussed. The GABA content of green coffee beans may serve as a potent tool to characterize the type of postharvest treatment applied in coffee processing.     

Accumulation of γ-aminobutyric acid in germinated soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) in relation to glutamate decarboxylase and diamine oxidase activity induced by additives under hypoxia

High levels of γ-aminobutyric acid (GABA) accumulate in plant tissues under various stresses and exogenous additives. The purpose of this research is to provide an effective finding that can prove a rapid accumulation of GABA in germinated soybean (Glycine max L.) in response to different additives under hypoxia. Hypoxia-induced GABA accumulation in soybean embryo resulted in part from polyamine oxidation. Response to different concentration of glutamate (Glu), pyridoxal phosphate, arginine, CuCl₂, NaCl, and CaCl₂, a significant difference including GABA accumulation, changes of Glutamate decarboxylase (GAD), and Diamine oxidase activity (DAO) activity in germinated soybean under hypoxia occurred (p < 0.05) and the maximum accumulation of GABA were 4.07, 3.02, 3.50, 3.26, 4.00, and 3.30 g kg⁻¹ DW respectively, which were significantly higher than those germinated soybean under normal culture (CK) and hypoxia culture (CK₀) (p < 0.05). The GAD and DAO have different distributions in cotyledon and embryo of germinated soybean, and the enzyme activity mainly located in embryo of germinated soybean. Germinated soybean is a good resource of GABA-rich food. Different additives have significant effects on GABA production, among which Glu and NaCl are ideal material for GABA accumulation.     

Study of nonenzymic browning in α-amino acid and γ-aminobutyric acid/sugar model systems

The reactivities, in nonenzymic browning, of γ-aminobutyric acid (GABA), a non-protein amino acid with wide natural occurrence and potential health benefits as it occurs in foods, and of the α-l-amino acids arginine, glutamic acid, glutamine, leucine, lysine, and phenylalanine were investigated by heating equimolar mixtures of glucose and the cited amino acids at 110 °C at pH 6.0 for different times (0–4 h). Linear regression analysis indicated that the colour development in a GABA/glucose mixture was slower than that of a lysine/glucose mixture and comparable to that of a phenylalanine/glucose mixture. High-performance anion-exchange chromatography (HPAEC) with integrated pulsed amperometric detection (IPAD) showed that the decrease in GABA levels (ca. 10% after heating for 4 h) as a function of heating time was smaller than that of glucose (ca. 30% after heating for 4 h). At the same time, glucose to fructose isomerisation took place. After 20 min of heating at pH 6.0, all mixtures showed a fructose peak, the area of which increased with heating time. However, after correcting for fructose isomerisation, glucose losses were still higher than amino acid losses. In contrast to its precursor glutamic acid, GABA was stable during heating of a solution containing it alone. Heating of GABA-containing d-sugar solutions (xylose, fructose, glucose, maltose and sucrose) showed that the relative order of colour yield was pentose > hexose > disaccharides. As well as glucose to fructose isomerisation, HPAEC–IPAD allowed monitoring of the different isomerisation reactions occurring, and also disaccharide hydrolysis in the different GABA/sugar mixtures.     

Contents of lovastatin, γ-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia

Lovastatin, γ-aminobutyric acid (GABA) and ergothioneine were analysed in mushrooms. Among fruiting bodies, Pleurotus ostreatus (Japan) and Agaricus bisporus contained the highest amount of lovastatin (606.5 and 565.4 mg/kg, respectively) whereas among mycelia, Cordyceps sinensis and Antrodia salmonea contained the highest (1365 and 1032 mg/kg, respectively). Among fruiting bodies, Flammulina velutipes and Boletus edulis contained the highest amount of GABA (229.7 and 202.1 mg/kg, respectively) whereas among mycelia, Cordyceps cicadae, C. sinensis and Agaricus blazei contained the highest (254.9, 220.5 and 200.4 mg/kg, respectively). Among fruiting bodies, Pleurotus citrinopileatus, P. ostreatus (Korea), P. ostreatus (Taiwan) and Pleurotus salmoneostramineus contained the highest amount of ergothioneine (2850.7, 1829.4, 1458.4 and 1245.0 mg/kg, respectively) whereas among mycelia, Pleurotus eryngii contained the highest (1514.6 mg/kg). Ergothioneine was detected in all samples. However, the Pleurotus genus contained considerably high amount of ergothioneine. Overall, these results might be related to their beneficial effects.     

Antihypertensive effect of mulberry leaf aqueous extract containing γ-aminobutyric acid in spontaneously hypertensive rats

We hypothesised that mulberry leaves (ML) exert their antihypertensive effect through γ-aminobutyric acid (GABA) and that the antihypertensive activity of ML extract would be comparable to GABA alone in spontaneously hypertensive rats (SHR). We found that single administration of a water extract from leaves of Morus alba L. (WEML) lowered systolic blood pressure (SBP) transiently in a dose-dependent manner. Interestingly, the SBP-lowering effect of WEML was significantly higher than after treatment with GABA alone. We further found that WEML strongly inhibited angiotensin I-converting enzyme (ACE) activity in vitro with an IC₅₀ value of 29.8 mg/ml. These results suggest that WEML has a transient antihypertensive effect in vivo that may involve a mechanism of ACE inhibition in addition to GABA.     

Effects of rumen-protected γ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows

This experiment was conducted to investigate the effects of rumen-protected γ-aminobutyric acid (GABA) on performance and nutrient digestibility in heat-stressed dairy cows. Sixty Holstein dairy cows (141 ± 15 d in milk, 35.9 ± 4.3 kg of milk/d, and parity 2.0 ± 1.1) were randomly assigned to 1 of 4 treatments according to a completely randomized block design. Treatments consisted of 0 (control), 40, 80, or 120 mg of true GABA/kg of dry matter (DM). The trial lasted 10 wk. The average temperature-humidity indices at 0700, 1400, and 2200 h were 78.4, 80.2, and 78.7, respectively. Rectal temperatures decreased linearly at 0700, 1400, and 2200 h with increasing GABA concentration. Supplementation of GABA had no effect on respiration rates at any time point. Dry matter intake, energy-corrected milk, 4% fat-corrected milk, and milk fat yield tended to increase linearly with increasing GABA concentration. Supplementation of GABA affected, in a quadratic manner, milk protein and lactose concentrations, and milk protein yield, and the peak values were reached at a dose of 40 mg of GABA/kg. Milk urea nitrogen concentration responded quadratically. Total solids content increased linearly with increasing GABA concentration. Supplementation of GABA had no effect on milk yield, lactose production, total solids, milk fat concentration, somatic cell score, or feed efficiency. Apparent total-tract digestibilities of DM, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber were similar among treatments. These results indicate that rumen-protected GABA supplementation to dairy cows can alleviate heat stress by reducing rectal temperature, increase DM intake and milk production, and improve milk composition. The appropriate supplemental GABA level for heat-stressed dairy cows is 40 mg/kg of DM.     

The multi-step process optimisation of candied kidney beans with high nutrients and γ-aminobutyric acid retention

The candied kidney bean contains a vital bioactive compound called γ-aminobutyric acid (GABA), which could be easily lost in the traditional producing processes like soaking, cooking and candying. The traditional procedure distinctly decreased the GABA content to the GABA retention rate of 34.01% in the candied kidney bean. In this study, the optimum technological methods of soaking, cooking and candying were determined by single factor experiment according to the GABA content. The optimised producing condition was that the kidney bean was soaked at 55°C for 5 h, followed by atmospheric steaming for 35 min and then subjected to microwaving candying at 150 W for 25 min. By the optimal procedure, the GABA content retention reached 75.33%. Meanwhile, the texture, nutrient contents and antioxidant capacity were obviously improved in the candied kidney beans produced by the optimal methods.     

Effects of steeping and anaerobic treatment on GABA (γ-aminobutyric acid) content in germinated waxy hull-less barley

Effects of steeping conditions (time, temperature and soaking solution) and anaerobic storage on the gamma-aminobutyric acid (GABA) content in waxy hull-less barley grains during germination was examined. The barley kernel was steeped for 16 h at different temperatures (5, 15 or 35 °C) either in water or in a buffer solution (pH 6.0, 50 mmol/L sodium acetate) and then germinated at 15 °C for 72 h. To reach the optimum water content (36–44 g/100 g) for germination, a longer steeping period was required when steeping temperature was lower (16 h at 5 °C vs. 8 h at 15 °C). At 35 °C for steeping, however, the water content in the grains increased excessively, and thus germination percentage became much less than those at 5 and 15 °C. The GABA content increased with increasing germination time and was higher in the buffer solution than water. These findings indicate that the glutamate decarboxylase (GAD), which is the rate-limiting enzyme for GABA synthesis, is more activated by extending germination at controlled pH (6.0). An anaerobic storage with nitrogen in the dark for the germinated barley grains substantially raised the GABA content: 14.3 mg/100 g after the treatment for 12 h, which was four times higher than that of control sample (3.7 mg/100 g). Overall results suggest that the steeping prior to germination greatly affects the GABA production during the germination of barley, and the anoxia storage with nitrogen after the germination increases the GABA content.     

Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that α_S2-casein, β-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.     

Effects of components in culture medium on glutamate decarboxylase activity and γ-aminobutyric acid accumulation in foxtail millet (Setaria italica L.) during germination

The effects of glutamic acid (Glu), pyridoxal-5-phosphate (PLP) and calcium chloride (CaCl₂) in culture medium on glutamate decarboxylase (GAD) activity and γ-aminobutyric acid (GABA) accumulation in foxtail millet (Setaria italica L.) during germination were investigated in this study. The components in culture medium for GABA accumulation were optimised using response surface methodology (RSM). Results showed that GAD activity and GABA yield were dependent on the addition of Glu, PLP and CaCl₂ into the culture medium. Box–Behnken design indicated that the optimal culture components for GABA accumulation were: Glu at a concentration of 1.2 mg/ml, PLP at a concentration of 50 μM and CaCl₂ at a concentration of 2.5 mM. Under the optimal conditions, the maximal production of GABA (42.9 mg/100 g FW) was obtained. Analysis of variance for the regression model suggested that the model can quite exactly predict GABA accumulation in millet during germination.     

Changes in γ-aminobutyric acid content and related enzyme activities in Jindou 25 soybean (Glycine max L.) seeds during germination

In order to accumulate γ-aminobutyric acid (GABA), soybean seeds (cultivar Jindou 25) were germinated for 102 h at different temperatures (19, 25 and 32 °C). The content of GABA, glutamic acid and the activity of the glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) in soybeans during germination were investigated. The results showed that the germination temperature and germination time had great influences on GABA content and the related enzyme activities in soybean seeds. As compared to raw soybeans, an increase in the content of GABA and glutamic acid was observed, as well as GAD activity in soybeans during germination, while germination at 32 °C was better for accumulating GABA in soybeans. The GABA-T activity first decreased and then increased at 19 °C and 25 °C, on the contrary, it first increased and then decreased sharply during germination at 32 °C. These results indicate that the increase of GABA content can be attributed to the changes of GAD and GABA-T activities rather than enough glutamic acid resulting from the degradation of protein during germination of Jindou 25 seeds. However, more assays need to be further performed with more soybean cultivars.